Subclass I of the receptor tyrosine kinase (RTK) super family consists of ErbB receptors and comprises four members: EGFR/ErbB1, HER2/ErbB2, ErbB3 and ErbB4. All members have an extracellular ligand-binding region, a single membrane-spanning region and a cytoplasmic tyrosine-kinase-containing domain. Tyrosine kinases are a class of enzymes that catalyze the transfer of the terminal phosphate of adenosine triphosphate to tyrosine residues in protein substrates.
The ErbB receptors are expressed in various tissues of epithelial, mesenchymal and neuronal origin. Under normal conditions, activation of the ErbB receptors is controlled by the spatial and temporal expression of their ligands, which are members of the EGF family of growth factors. Ligand binding to ErbB receptors induces the formation of receptor homo- and heterodimers and activation of the intrinsic kinase domain, resulting in phosphorylation on specific tyrosine kinase residues within the cytoplasmic tail. These phosphorylated residues serve as docking sites for various proteins, the recruitment of which leads to the activation of intracellular signaling pathways.
EGFR and HER2 are known to play an essential role in regulating cell proliferation and differentiation. They have a strong tendency to assemble with other HER receptors into homo- and/or heterodimers upon extracellular growth factor binding, which results in various forms of signal transduction pathways activation, leading to either apoptosis, survival, or cell proliferation. Mounting evidence suggests that not only receptor expression or receptor overexpression but also communication among HER receptors plays a crucial role in tumor behavior (Kumagai et al., 2001, PNAS 98, 5526). In particular, binding of receptor-specific ligands to the ectodomain of EGFR often results in the recruitement of HER2, as the preferred heterodimerization partner (e.g. Klapper et al., 1999, PNAS 96, 4995). As HER2 is the only HER family member that does not bind a known specific ligand, its principal biological function, as a signal transducer, appears to result from its participation in heterodimeric receptor complexes with EGFR or other HER receptors (e.g. Konecny et al., 2006, Cancer Res. 96, 1630).
Recent studies (J. Schlessinger, 2002, Cell 110, 669) show that receptor dimerization is mediated by receptor-receptor interactions in which a loop protruding from neighboring receptors mediates receptor dimerization and activation. Receptor dimerization is essential for stimulating of the intrinsic catalytic activity and for the self-phosphorylation of growth factor receptors on tyrosine residues.
It should be remarked that receptor protein tyrosine kinases are able to undergo both homo- and heterodimerization, wherein homodimeric receptor combinations are less mitogenic and transforming (no or weak initiation of signaling) than the corresponding heterodimeric combinations. Heterodimers containing ErbB2 are the most potent complexes (Yarden and Sliwkowski, 2001, Nature Reviews, Molecular cell Biology, volume 2, 127-137; Tzahar and Yarden, 1998, BBA 1377, M25-M37).
Two important types of ErbB inhibitor are in clinical use: chimeric, humanized or fully human antibodies directed against the extracellular domain of EGFR or ErbB2, and small-molecule tyrosine-kinase inhibitors (TKIs) that compete with the ATP in the tyrosine-kinase domain of the receptor.
The use of mAbs binding with high affinity to these two members of the ErbB (HER) family, EGFR and HER2, thus appears to be rational for the development of new cancer therapy strategies which would have the potential to inhibit the receptor dimerization. Until now, however, each of the two mAbs have been used in cancer therapy individually in conjunction with various chemotherapeutic drugs (Slamon et al., 2001, New Engl. J. Med 344, 783; Baselga et al., 2000, J Clin Oncol. 18, 904) or more recently in association with different drugs having tyrosine kinase receptor inhibition properties (e.g. Normanno et al., 2002, Ann Oncol 13, 65) The action of small tyrosine kinase inhibitor molecules, however, cannot be compared with the potential biological activity induced by the binding of a high molecular weight antibody molecule.
The mechanism of anti-tumor activity of individual anti-ErbB/HER receptor mAbs is not entirely understood. A series of experimental results in mice KO for the Fc receptor strongly suggested that most of the anti-tumor effect was due to the recruitment of effector NK cells by an antibody dependent cell-mediated cytotoxic (ADCC) mechanism. However, the evidence of inhibition of receptor phosphorylation and receptor internalization induced on tumor cells by the anti-ErbB/HER receptor mAbs argues in favor of an apoptotic or cytostatic signal transduced through the receptor (e.g. Friedman et al., 2005, PNAS 102, 1915).
There are several anti-HER/ErbB antibodies in clinical studies and already approved and on market, for example matuzumab, cetuximab, panitumumab and trastuzumab.
Humanized monoclonal antibody 425, also designated as matuzumab (hMAb 425, U.S. Pat. No. 5,558,864; EP 0531 472) and chimeric monoclonal antibody 225 (cMAb 225), both directed to the EGF receptor, have shown their efficacy in clinical trials.
The c225 antibody (cetuximab, ERBITUX®) was demonstrated to inhibit EGF-mediated tumor cell growth in vitro and to inhibit human colorectal tumors in vivo received marked approval in 2003. The antibody as well as in general all anti-EGFR antibodies, appear to act, above all, in synergy with certain chemotherapeutic agents (i.e., doxorubicin, adriamycin, taxol, and cisplatin) to eradicate human tumors in vivo in xenograft mouse models (e.g. EP 0667165). Furthermore, it could be shown that the combination of the anti-EGFR antibody c225 with the second anti-EGFR antibody matuzumab shows a synergistic effect in vitro models, indicating that these two antibodies directed to the same receptor bind to different epitopes of EGFR (WO 2004/032960).
The mouse antibody 4D5 directed to the HER2/ErbB2 was further found to sensitize ErbB2-expressing breast tumor cell lines to the cytotoxic effects of TNFα (U.S. Pat. No. 5,677,171). A recombinant humanized version, designated as huMAb4D5-8, rhuMAb HER2, trastuzumab, or HERCEPTIN® (U.S. Pat. No. 5,821,337) is clinically active in patients with ErbB2-overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy (Baselga et al., J. Clin. Oncol. 14:737-744 (1996)). HERCEPTIN® received marketing approval in 1998 for the treatment of patients with metastatic breast cancer whose tumors overexpress the ErbB2 protein.
Although, as stated above, the therapeutic efficacy trastuzumab in breast carcinoma is well demonstrated, it is strictly limited and only approved for 30% of breast cancer patients whose tumor overexpress HER2. 70% of the breast cancer patients do not or insufficiently respond to trastuzumab because their individual tumor do not overexpress or do not sufficiently express HER2. In other cancers and or individual cancers, HER2 is overexpressed in a significant percentage of cases ranging from 43-69%, whereas EGFR is usually overexpressed in a range from 45-95%. However, as a rule, the levels of HER2 expression are in principal low in the majority of tumors. Furthermore, the overexpression of EGFR and HER2 receptors is often caused by encoding gene amplification (Hynes et al., 2005, Nat Rev Cancer 5, 341). Thus, the present day consensus is that anti-HER2 mAb is inefficient in tumors with low HER2 expression or missing overexpression, which is the case, for example, for most pancreatic carcinoma in the clinic (e.g. Saxby et al., 2005, J Surg Pathol 29, 1125).
Panitumumab (VECTIBIX®) is a fully human anti-EGFR antibody and has recently received marked approval in the US (Schwartz et al., 2002, Proc. Am Soc Clin Oncol 21, 24).
Adenocarcinomas of the pancreas remain one of the most difficult malignancies to treat. The incidence has steadily increased over the past four decades, and its prognosis is still dismal, despite tremendous efforts in early diagnosis and therapy. At the time of diagnosis, the majority of patients (80-90%) have locally or metastatic tumors. Even with a complete surgical resection, the five-year survival rate is less than 20% (Wagner et al., Br J Surg 2004; 91: 586-94). The conventional therapy associating both surgery and radiotherapy alone or in combination with chemotherapy shows modest efficacy in local control and palliation, and no real progress in patient survival (Azria et al., Bull Cancer 2002; 89: 369-79, Azria et al., Pancreas 2002; 25: 360-5, Li et al. Lancet 2004; 363:1049-57.2-4). Accordingly, novel approaches to human pancreatic carcinoma therapy are urgently needed.
It is well documented that pancreatic adenocarcinomas and displasias frequently overexpress tyrosine kinase receptors. Overexpression of EGFR in pancreatic cancer is associated with advanced disease at presentation and reduced median survival time. The significance of HER2 expression/overexpression in pancreatic cancer prognosis is not as clear. Indeed, no correlation between tumor differentiation degree and the level of HER2 expression in human pancreatic specimens has been reported (Dugan et al., Pancreas 1997; 14: 229-36). This might be explained by recalling that the level of HER2 does not mediate mitogenesis by itself and that heterodimerisation has to be activated.
The concept of blocking EGFR and HER2 is in principal known and has been applied in other models expressing high levels of EGFR and/or HER2. An additive but not an synergistic effect on reducing cell proliferation in vitro was shown using combined treatment either with chimeric anti-EGFR antibody mAb c225 (cetuximab) and humanized anti-HER2 antibody 4D5 (trastuzumab) on the human ovarian cancer cell line OVCAR 420 (Ye et al. 1999, Oncogene 18, 731) or with trastuzumab and the murine variant of mAb 225 on EGFR-dependent colon cancer cell lines (Kuwada et al., Int J Cancer 2004; 109: 291-301).
Furthermore, different groups addressed the simultaneous attack of HER1 and HER2 by associating the chemical EGFR kinase inhibitor ZD1839 (Iressa) with trastuzumab (Normanno et al., Ann Oncol 2002; 13: 65-72) and found that in the cell lines SK-BR-3 and BT-474 the combination of these latter compounds induces a better anti-proliferative effect than the two compounds used separately, particularly in terms of induction of apoptosis.
However, little information is available concerning antibody efficacy and the actual function of EGFR and HER2 receptors in specific cancer tissue expressing low levels of at least one of these receptors, for example, pancreatic cancer, targeted by specific anti-ErbB antibodies, and up to date no clinical studies have evaluated the efficacy of targeting concurrently these two receptors in these tumors with suitable monoclonal antibodies.
Based on the lack of an effective therapy and implication of EGFR and HER2 in specific cancer expressing low levels of at least one of these two types of receptor, there is a motivation of blocking EGFR and HER2 simultaneously in a cancer specific model in which preferably a high EGFR expression and no significant or low-level expression is presented, wherein said expression pattern is evaluated by immunocytochemical analysis or by using flow cytometry analysis technique.